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January2004 Vol.41 Issue:      1 Table of Contents
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Does Parkinson's disease have an immunological basis ? The evidence and its therapeutic implications

Shereen Zakarya1, Elham R. Abd El Sameea2, Azza Abd El Baky2, Noha El Mashed2
Departments of Neurology1, Clinical Pathology2, Mansoura University

ABSTRACT

Objectives: This study aimed to clarify the role of some cytokines in the aetieopathogenesis of Parkinson's Disease (PD). Subjects: Thirty patients with Parkinson's Disease (19 males and 11 females) with ages ranged from 45 to 77 years (mean 65.40±7.44) and 20 healthy subjects of matched age and sex as a control group were enrolled in the study. The patients, were selected from the Neurology Outpatient Clinic of Mansoura  University Hospital. Methods: All patients were subjected to complete  neurological examinations together with unified Parkinson's Disease Rating Scale (UPDRS) and Modified Hoehn and Yahr Staging scale. Serum IL-Ib, IL 6, IL-8 and TNF-a were determined for all patients and control group. Results: There was significant increase of all studied cytokines (P<0.001) in patients compared to control group. There was no statistical significance correlation between cytokine level and duration of illness nor therapy with levodopa (P>0.05). But the study showed significant statistical correlation (P<0.001) between TNF-a & IL-6 serum levels and severity of the disease detected by UPDRS and modified Hoehn and Yahr Staging. Conclusion: The immunological system is involved in the pathogenesis of PD. The Data suggest that a mixture of cytokines may help induce and perpetuate the inflammatory process. So anti-inflammatory such as cytokines blocking agents may be an important element in the elaboration of new therapies for PD.

(Egypt J. Neurol. Psychiat. Neurosurg., 2004, 41(1): 273-282).

 




INTRODUCTION

 

More than 180 years ago, James Pakinson first described the disorder that bears his name. Parkinson's Disease (PD) is a neurodegenerative disorder of unknown cause that affect over I million people in North America1. The aetiology of PD and its progressive nature are incompletely understood and are most likely multifactorial. The discovery of mutations in alpha synuclein, parkin, and ubiquitin C terminal hydrolase L1 (UCHL–1), associated with hereditary forms of parkinsonism2. Several other processes in PD, including oxidative stress, immune/inflammatory process, Mitochondrial dysfunction, excitotoxicity, trophic factors deficiency and finally apoptosis. Environmental insults may trigger or facilitate neurodegeneration.4 

There is increasing evidence that underlying peripheral immune/inflammatory process contribute to PD pathogenesis5,6. There is an increase in reactive microglia in the striatum and substantia nigra of patients with idiopathic PD. The activated microglia express the apoptotic cytokine, tumour necrosis factor [alpha] (TNF-a), interleukin -6 (IL–6) and the third member of the "inflammatory triad", interleukin-1 beta (IL-1b)7,8.

The aim of this work is to clarify the role of some proinflammatory cytokines as IL-1b, IL-6, IL-8 and TNF-a and the aetieopathogenesis and performance of Parkinson's Disease.

 

SUBJECTS AND METHODS

 

Subjects

The present study was conducted on thirty patients suffering from Parkinson's disease. They were selected from the Neurology Outpatient Clinic of Mansoura University Hospital (19 males and 11 females) with ages ranged from 45 to 77 years (Mean 65.40±7.44)

Twenty healthy subjects of matched age and sex were selected as a control group.

All patients and controls were non smokers or stop smoking since at least 6 months.

All patients included in the study fulfill the Diagnostic Criteria of Idiopathic PD which are: Resting tremors, rigidity, bradykinesia, flexed posture, loss of postural reflexes and freezing phenomenon. At least 2 of these features, with at least one being either tremors at rest or bradykinesia must be present.

Patients with history of strokes, head injuries, encephalitis, oculogyric crisis, drug abuse or neuroleptic or antipsychotic medications, supra-nuclear gaze palsy, cerebellar signs and early severe dementia were excluded from the study.

 

Methods

Patients and controls were assessed using the following battery of investigations:

*      Thorough Medical and Neurological examinations.

*      Unified Parkinson Disease Rating Scale (UPDRS): UPDRS is rating tool to follow the longitudinal course of Parkinson's Disease. It is made up of the [1] Mentation, Behaviour, and Mood, [2] Activities in Daily living [3] motor sections and [4] complications of therapy. These were evaluated by interview. Some sections require multiple grades assigned to each extremity. A total of 199 points are possible. 199 represents the worst (total disability), O – no disability.

*              Modified Hohen & Yahr Staging

Stage O:  No signs of the disease

Stage l :   Unilateral disease.

Stage 1.5:   Unilateral plus axial involvement

Stage 2:   Bilateral disease without impairment of balance.

Stage 2.5:   Mild bilateral disease with recovery on pull test.

Stage 3:   Mild or Moderate bilateral disease; some postural instability

Stage 4:   Severe disability; still able to walk or stand unassisted.

Stage 5:   Wheelchair-bound or bed-ridden unless aided.

*              Laboratory investigations.

1.    Routine investigations:

- Liver function tests

- Kidney function tests

- Complete Blood picture

2.    Immunological assay:

Five ml venous blood  samples were withdrawn. Serum was separated rapidly after being clotted and stored at - 70°C until the time of cytokine assay (IL-Ib, IL-6, IL8, TNF-a)

Principle of the assay

-    Serum IL-Ib and IL 6 were assayed by a solid phase sandwich enzyme linked immune assay (ELISA) using commercial kits (Diaclone, France). A specific monoclonal antibody has been coated into wells of the microtiter strips provided. Samples, including standards of known concent-ration, control specimens and unknown samples are pipetted into these wells. During the first incubation the IL-1b, IL-6 antigens and a specific bio-tinylated monoclonal antibodies are simultaneously incubated. After washing, the enzyme (streptavidin – peroxidase) is added. After incubation and washing to remove all the unbound enzyme, a substrate solution which is acting on the bound enzyme is added to induce a coloured reaction product, Its intensity is directly proportional to the concentration of the cytokine present in the samples.

-    Serum IL-8 was assayed using the quantitative sandwich enzyme immunoassay technique (Quantikine RTM & D systems) and Serum level of TNF-a were assayed using commercially available enzyme linked immunosorbent assay (ELISA) kit [TNF-a, ELISA, Biosource, Europe S.A., Belgium] according to manufacturer's instructions.

 

Statistical Methods

            Results were evaluated and data were compared using IBM computer. The statistical analysis was done using SPSS (statistical programmes of social science) programme version 10.

 

The student T test was used for comparing means of quantitative data. Person correlation coefficient (r) was used for correlation.

 

A (P) value of 0.05 was considered the limit below which the difference of the values would be statistically significant. A (P) value of 0.01 was considered the limit below which the difference of the values would be highly statistically significant.

 

RESULTS

 

Results were shown in tables (1–6) and Figs. (1–2).

*     There was highly significant increase of serum IL-1b, IL-6, IL-8, TNF­-a (P<0.001) in patients with Parkinson's Disease when compared with control group (Table 1 & Figs. 1-2)

*     IL-1b and IL-6 showed significant increase with age, while IL 8 & TNFa didn't show any increase with age (Table 2).

*     No correlation between IL-1b, IL-6, IL-8 & TNF-a  and disease duration (Table 3).

*     Weak positive correlation between IL-1b, IL-6, IL-8 & TNF-a  and levodopa therapy with non significant statistical difference (P>0.05) (Table 4).

*     Positive correlation between TNF-a & IL-6 and disease severity detected by (UPDRS and Modified Hoehn and Yahr) with significant statistical difference (P<0.001) (Tables 5 and 6).


 

 

 

Table 1. IL-Ib, IL-6, IL-8 & TNFa in patients versus control group.

 

Group

 

Parameter

Patients

Control

P value

Mean ± S. D

Mean ± S. D

IL-Ib (pg/ ml)

247.2 ± 51.6

105.85 ± 41.06

P< 0.001

IL-6 (pg / ml)

2.72 ± 1.24

1.36 ± 0.30

P< 0.001

IL-8 (pg / ml)

42.43 ± 6.16

20.65 ± 4.17

P < 0.001

TNF-a  (pg / ml)

54.07 ± 3.57

10 ± 3.26

P < 0.001

 

 

 

Table 2. Serum level of IL-Ib, IL-6, IL-8 and TNF-a relation to age.

 

Group

 

 

Parameter

< 65

n = 11

> 65

n =19

P value

IL-Ib (mean ± S.D)

180.27  ± 49.61

257.58

P < 0.05

IL-6 (mean ± S.D)

1.69  ± 1.07

2.84  ± 1.35

P < 0.05

IL-8  (mean ± S.D)

46.09  ± 4.06

40.32  ± 6.25

P > 0.05

TNF-a  (mean ± S.D)

54.18  ± 3.46

54  ± 3.73

P > 0.05

 

 

 

Table 3. Correlation coefficient between IL Ib, IL6, IL8, TNFa and disease duration.

 

     Parameter

Duration

IL-1b

IL-6

IL-8

TNF [a]

r

P

r

P

r

p

r

p

Duration of illness

0.235

>0.05

0.159

>0.05

0.126

>0.05

0.331

>0.05

 

 

 

Table 4. Relation between ILIb, IL 6, IL8, and TNF-a & levodopa therapy.

 

Group

 

Parameter

Group I

(treated with levodopa)

n = 16

Group II

(non treated with levodopa)

n = 14

P value

IL-Ib

239.38  ± 43.99

246.14 ± 59.53

P > 0.05

IL-6

2.89  ± 1.30

2.52  ± 1.18

P > 0.05

IL-8

40.63 ± 4.75

41.07 ± 7.41

P > 0.05

TNF-a

54.81  ± 4.19

54.36  ± 2.82

P > 0.05

 

Table 5. Correlation coefficient between serum IL-Ib & IL-6 & IL-8 and TNF-a and Unified Parkinson's Disease Rating Scale (UPDRS).

 

Group

 

Parameter

UPDRS

Total score

Rigidity score

Tremor score

IL-Ib 

r

P

10.181

> 0.05

10.034

> 0.05

10.110

> 0.05

IL-6

r

P

0.411

< 0.001

0.198

< 0.001

0.120

< 0.001

IL-8

r

P

0.184

> 0.05

0.240

> 0.05

0.325

> 0.05

TNF-a

r

P

0.406

< 0.001

0.591

< 0.001

0.665

< 0.001

Table 6. Correlation coefficient between serum IL-Ib &  IL-6, IL-8, TNF-a and Modified Hoehn and Yahr Staging.

 

Group

Parameter

Modified Hoehn and Yahr Staging

R

P

IL-Ib

0.111

> 0.05

IL-6

0.152

< 0.001

IL-8

0.309

> 0.05

TNF-a

0.543

< 0.001

 

 

 

Fig. (1): Level of IL-1b in patients and controls.

 

Fig. (2): Level of IL-6 in patients and controls.

 

 


DISCUSSION

 

Proinflammatory cytokines is implicated in the pathogenesis of idiopathic parkinsonism9. Although the brain has long been considered "immunoprivileged", the products of a remote or systemic immune process can access or communicate with, its immune system10. The central action of cytokines act through specific receptors located on the dopaminergic neurons which contain intracytoplasmic death domains and are involved in apoptosis11. Alternatively, the cytokines are known to induce the expression of nitric oxide via the induction and activation of the low affinity Ig E receptor called CD23. The gradual increase of nitiric oxide from the glial cells may account for the increased oxidative stress12. The activated microglia also produce COX-2, which produces free radicals as a by product of prostaglandin synthesis13.

IL–1b is a cytokine with multiple proinflammatory and cell growth modulatory actions14, It is produced predominantly by activated macrophages, and also by astrocytes, fibroblasts and neurons its influence on the inflammatory cascade includes the induction of prostaglandins, collagenase, phospholipase A2, chemotactic cytokines such as IL-8 and other inflammatory cytokines such as TNF-a, IL 6 and colony- stimulating factors.15,16

The present study showed significant increase of serum IL-1b in parkinsonian patients (mean 247.2±51.6 pg/ml) when compared with control group (mean 105.85±41.06 pg/ml) p≤0.001. This go well with those obtained by other studies.15,17 (Table 1) (Fig. 1)

Moreover IL-1b showed significant increase with age (mean 257.58±51.114 pg/ml) in patients aged more than 65 years. The same results were obtained by Barbeau18, who concluded that IL-1b increases progressively with age in parkinsonian patients when compared with control group in the same age (Table 2).

IL-6 is a pleitropic cytokine with both proinflammatory19 and anti-inflammatory effects20. Its expression in association with TNF-a  and IL-1b has been well described in the sitting of tissue injury21. IL-6 showed significant increase in patients with Parkinson's Disease (mean 2.72±1.24 pg/ml) when compared with control group (mean 1.36±0.30 pg/ml) p≤0.001.

Our results go in hand with Rothwell et al16 who found significant increase of plasma levels of IL-6 in parkinsonian patients. IL-6 also showed significant increase with age. This is in accordance with critchley22 (Tables 1, 2 and Fig. 2).

IL 8 is chemotactic cytokine23. Poupard et al.24, found significant increase of plasma levels of IL 8 in parkinsonian patients. Fiszer et al.25, didn't find any measurable levels of plasma IL-8 in patients with Parkinson's Disease. Our results showed significant increases of plasma levels of IL-8 in parkinsonian patients. The differences of values may be explained by difference of sampling time from the onset of the disease or the severity of the disease (Table 1).

TNF-a which is a potent proinflammatory cytokine and produced by activated macrophages, has various biological and cytotoxic effects. TNF-a is of importance because it can provoke cell death26. In our study, TNF-a serum levels showed significant increase in parkinsonian patients than controls (mean 54.07±3.57 pg/ml, 10±3.26 pg/ml respectively) p≤0.001. Dobbs et al.27, concluded that TNF-a is the most proinflammatory cytokine increases in Parkinson's Disease (Table 1).

Our results showed that there was no correlation between serum levels of IL-Ib, IL- 6, IL-8, TNF-a with disease duration (Table 3).

This is in accordance with Hirch et al.28, who reported that serum levels of the studied cytokines did not display any correlation with disease duration in parkinsonian patients.

Also, in the present study, there was no significant correlation between the studied cytokine levels and levodopa therapy. This go well with those obtained by other authors29 (Table 4).

Regarding the relation between the studied cytokine levels and disease severity determined by UPDRS and modified Hoehn and Yahr staging, TNF-a and IL-6 showed significant positive correlation (p<0.001) with the disease severity. Charlett et al.30, concluded that circulating TNF-a may be a biochemical predictor, or indeed mediator of the severity of the disease. Also, Dobbs et al.27 concluded that TNF-a and IL-6 appeared elevated in parkinsonians whose postural and psytchomotor responses were abnormal (Tables 5 and 6).

From this study we can conclude that the immunological system is involved in the pathogenesis of PD. However, we are not able to determine weather the disturbances described above constitute a primary or secondary phenomenon in PD. The data suggest that a mixture of cytokines may help induce and perpetuate the inflammatory process. So anticytokine therapy may be an important element in the elaboration of new therapies for PD, which may modulate the inflammatory process.

Peroxisome proliferator activated receptor (PPAR) [gamma] agonists such as troglitazone and pioglitazone are a member of the nuclear receptor superfamily that suppress the expression of the proinflammatory cytokines IL-Ib, TNF-a  and IL-6. It also inhibit nitric oxide and Cox-2 activity31,32.

Considerable interest has been shown in the potential utility of teteracycline derivative minocycline to reduce glial activation which is the main source of the proinflammatory cytokines28.

Various compounds such as non steroidal anti-inflammatory drugs (NSAIDs) with their targets COX, NF (Kappa) B, and others, including steroids, immunophilins, thalidomide, and phosphodiesterase IV inhibitors have been studied in the treatment of parkinsonism with variable results33,34.

Recently gene manipulation modulate COX-2 and iNOS are of particular interest in such strategies33.

 

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7.      Floyd, R.A. (1999): Antioxidanats, oxidative stress and degenerative neurological disorders. Proc. Soc. Exp. Biol. Med. 222: 236 – 245.

8.      Raivich, G., L.L. Jones, A. Werner, et al., (1999): Molecular signals for glial activation: Pro- and anti – inflammatory cytokines in the injured brain. Acta Neurochir. Suppl. 73: 21:30.

9.      Kuhn W, Muller T, Nastos I, et al., (1997): The neuroimmune hypothesisi in Parkinson's disease. Rev Neurosci; 8: 29-34.

10.    Marttila RJ, Eskola J, Paivarinta M, et al. (1984): Immune functions in Parkinson's disease. Adv. Neurol; 40: 315 – 23.

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24.    Poupard A, Emile J. (1993): Autoimmunity in Parkinson's disease. In: Hassler RG, Christ JF, eds. Adv Neurol 40. Parkinson – specific motor and mental disorders. Role of the pallidum: pathophysiological, biochemical and therapeutic aspects. Eur. J Immunol; 23: 2375 – 8.

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الملخص العربى

 

هل يوجد أسس مناعية لمرض باركينسون ؟ الإثباتات والتطبيق العلاجى

 

الهدف من هذا البحث هو توضيح دور بعض السيتوكينات فى مرض باركينسون وقد اشتمل هذا البحث على 30 مريضا بمرض باركينسون (19 ذكرا،11 أنثى) تراوحت أعمارهم ما بين 45 إلى 72 عاما بمتوسط عمرى 65.40±7.44 عاما بالإضافة إلى عشرون شخصا أصحاء يمثلون المجموعة الضابطة وقد تم اختيار المرضى من عيادة المخ والأعصاب بمستشفى المنصورة الجامعى.

وقد تم فحص المرضى إكلينيكيا كما تم قياس مستوى الإنترلوكين 1 بيتا والإنترلوكين 6 والإنترلوكين 8 ومعامل نخر الأورام ألفا فى مصل الدم للمرضى والمجموعة الضابطة.

وقد أظهرت النتائج وجود زيادة ذات دلالة إحصائية فى مستوى السيتوكينات فى المرضى عند مقارنتهم بالمجموعة الضابطة، لم يثبت وجود أى علاقة بين مستوى السيتوكينات ومدة المرض أو العلاج بعقار الليفودوبا الذى يستخدم فى علاج مرض باركينسون.

كما أثبتت الدراسة وجود علاقة إيجابية بين مستوى بعض السيتوكينات فى البلازما (معامل نخر الأورام ألفا وإنترلوكين 6) وشدة المرض.

نستنبط من هذه النتائج أن مرض باركينسون له أسس مناعية ولذلك فإن استعمال مضادات الالتهابات مثل مضادات السيتوكينات يمكن أن يقلل من تدمير الخلايا العصبية إذا استخدمت كعلاج فى مرحلة مبكرة من مرض باركينسون.

 



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